In order to analyze drug interactions in humans and human gene polymorphisms, recent attention has been focused on in vivo drug metabolism. Also in the development of new drugs, the importance of studying in vivo drug metabolism is widely recognized and efforts are made to elucidate phenomena related to the effectiveness or toxicity of drugs by monitoring qualitative and quantitative changes in the drugs administered in vivo.
Although drug metabolism occurs in many tissues in the body, the major metabolic site for most drugs is the liver in terms of its high activity and weight. In the metabolism of fat-soluble drugs, a particularly important role is played by cytochrome P450 (forming a superfamily, abbreviated as CYP) which is localized in microsomes and categorized as a so-called drug-metabolizing enzyme. The basic reaction mechanism of this enzyme involves drug oxidation reaction resulting from oxygen activation mediated by cytochrome P450 reduced by the action of NADPH-cytochrome P450 reductase, as well as drug reduction reaction due to a low oxidation-reduction potential of cytochrome P450. It is currently reported that 80% or more of the clinically used drugs are metabolized by P450. In particular, metabolites produced as a result of oxidation reaction (Phase I reaction) are further metabolized into more water-soluble metabolites through glucuronide conjugation, sulfate conjugation, amino acid conjugation, acetyl conjugation, glutathione conjugation or the like (Phase II), thereby facilitating their excretion into urine or bile.
This P450 metabolism study has been conventionally conducted using reversed-phase chromatography in which a hydrophobic porous silica gel whose surface is modified with octadecyl groups is used as a stationary phase and solutes are separated by continuously increasing the concentration of an organic solvent in a mobile phase. However, organic solvents and buffers used in conventional mobile phases provide eluates containing contaminants and are disadvantageous in that baseline stabilization and detection sensitivity are significantly decreased because the contaminants produce strong UV absorption. Moreover, a column is required to be washed and equilibrated with an initial eluent before being continuously provided for the next analysis, which constitutes an additional disadvantage of prolonged analysis time. Further, detailed separation conditions including the flow rate, ionic strength and pH of eluent as well as column temperature should be determined for each solute; for example, in a clinical setting or elsewhere, there has been a strong demand for a simple method which allows separation of individual solutes under a single condition.
In the current clinical setting, an antigen-antibody reaction-based immunoassay (TDX) is actually used as a method for simple evaluation of drug-metabolizing capacity. However, such a method permits the measurement of only one item and hence requires repeated measurements when a larger number of items should be measured. Under present circumstances, such a method is therefore cost consuming and involves complicated steps.